Aspects of this invention relate in general to analytic methods involving separation of moiety fragments of interest such as, for example, biopolymer fragments, from other moieties not of interest and from one another and subsequent detection of those moieties. Some aspects of the invention relate in particular to the enzymatic digestion of polypeptides including proteins for the purpose of conducting various analyses of polypeptide fragments.
The clinical diagnostic field has seen a broad expansion in recent years, both as to the variety of materials of interest that may be readily and accurately determined, as well as the methods for the determination. In clinical chemistry these materials may be present in body fluids in concentrations below 10−12 molar.
In recent years, techniques have been developed for the analysis or determination of organic compounds present in extremely small quantities or at very low concentrations. For example, by combining chromatographic techniques such as liquid chromatography with various detection means such as mass spectrometry, sensitivity in the detection of analytes is enhanced.
The goal of proteomics is to identify and quantitate some or all of the proteins expressed in a cell as a means of addressing the complexity of biological systems. Typically, analysis techniques are employed to generate proteome maps. Proteome maps of normal cells and diseased cells are compared to detect proteins that are up- or down-regulated during physiological responses to disease. These proteins are excised for identification and characterization, using such methods as mass fingerprinting and mass spectrometry.
Column separation-detector techniques such as, e.g., LC/MS or HPLC/MS, are used routinely in proteomic studies as a tool to identify unknown proteins. Enzymatic digestion of model protein is a necessary step in analytic processes such as, for example, the process of peptide mass finger printing (PMF) or peptide mapping by mass spectrometer (MS). The enzymatic digestion of proteins is, of course, well known. It results in the breaking up of the protein molecule into smaller fragments. It is a technique used, for example, in the determination of the amino acid sequence in proteins. The mechanism of such cleavage, and hence the precise constitution of the fragments, varies with the enzyme used and the conditions (e.g. time, temperature and pH) under which the digestion is effected.
At present, enzymatic digestion in many column separation-detector techniques is done mostly off-line. The digestion process is a time consuming task, ranging from two hours to overnight with several manual sample manipulation steps such as desalting, sample purification and concentration and then sample introduction into, for example, LC/MS or HPLC/MS. Perceivably, the digestion process limits the applicability of high-throughput PMF analysis of proteins to quickly search proteins as potential targets for drugs and biomarkers for disease on a proteome-wide scale. Additionally, it makes the unknown protein analysis of both qualitation and quantitation difficult because of off-line steps involved. Multi-step off-line processes of digestion, purification and concentration can result in significant loss of digested fragments or low recovery, thereby reducing the effectiveness of the overall process.
There remains a need to perform analytical and diagnostic assays for proteins and other biopolymers where the biopolymers are digested and biopolymer fragments are collected, separated or detected.